Mutation in the beta2 nicotinic acetycholine receptor subunit associated with nocturnal frontal lobe epilepsy

ABSTRACT

A point mutation in the β2 subunit of an nicotinic acetylcholine receptor providing a V287M transition is associated with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE).

TECHNICAL FIELD

The present invention relates to mutations in the nicotinicacetylcholine receptor which are associated with idiopathic epilepsiesin particular, with autosomal dominant nocturnal frontal lobe epilepsy(ADNFLE).

BACKGROUND ART

Epilepsies constitute a diverse collection of brain disorders thataffect about 3% of the population at some time in their lives (Annegers,1996). An epileptic seizure can be defined as an episodic change inbehaviour caused by the disordered firing of populations of neurons inthe central nervous system. This results in varying degrees ofinvoluntary muscle contraction and often a loss of consciousness.Epilepsy syndromes have been classified into more than 40 distinct typesbased upon characteristic symptoms, types of seizure, cause, age ofonset and EEG patterns (Commission on Classification and Terminology ofthe International League Against Epilepsy, 1989). However the singlefeature that is common to all syndromes is the persistent increase inneuronal excitability that is both occasionally and unpredictablyexpressed as a seizure.

A genetic contribution to the aetiology of epilepsy has been estimatedto be present in approximately 40% of affected individuals (Gardiner,2000). As epileptic seizures may be the end-point of a number ofmolecular aberrations that ultimately disturb neuronal synchrony, thegenetic basis for epilepsy is likely to be heterogeneous. There are over200 Mendelian diseases which include epilepsy as part of the phenotype.In these diseases, seizures are symptomatic of underlying neurologicalinvolvement such as disturbances in brain structure or function. Incontrast, there are also a number of “pure” epilepsy syndromes in whichepilepsy is the sole manifestation in the affected individuals. Theseare termed idiopathic and account for over 60% of all epilepsy cases.

Idiopathic epilepsies have been further divided into partial andgeneralized sub-types. Partial (focal or local) epileptic fits arisefrom localized cortical discharges, so that only certain groups ofmuscles are involved and consciousness may be retained (Sutton, 1990).However, in generalized epilepsy, EEG discharge shows no focus such thatall subcortical regions of the brain are involved. Although theobservation that generalized epilepsies are frequently inherited isunderstandable, the mechanism by which genetic defects, presumablyexpressed constitutively in the brain, give rise to partial seizures isless clear. Certainly the study and isolation of the genes involved inrare families with primarily monogenic aetiology will aid inunderstanding the types of genes involved and the mechanisms of thedisease process in general.

One such form of idiopathic partial epilepsy inherited in a simpleMendelian manner is called autosomal. dominant nocturnal frontal lobeepilepsy (ADNFLE). This epilepsy, first described in 1994 (Scheffer etal., 1994) is characterized by clusters of frontal lobe motor seizuresoccurring during sleep, with onset usually occurring in childhood. Thecondition is clinically distinctive and relatively homogeneous, althoughseizure severity and specific frontal lobe seizure manifestations varywithin families (Hayman et al., 1997). Misdiagnosis as nightmares, nightterrors, hysteria, sleep paralysis, other parasomnias, or evenpsychiatric disorders is common if clinicians are unaware of ADNFLE.

Linkage analysis of a large family with this form of epilepsy identifieda locus on chromosome 20q13.2 most likely to contain the responsiblegene (Phillips et al., 1995). Using a positional candidate approach, theneuronal nicotinic acetylcholine receptor α4 subunit (CHRNA4) was anideal candidate gene due to the involvement of the nicotinicacetylcholine receptor (nAChR) in chemo-electrical transduction atcholinergic synapses of the central nervous system (CNS) and the factthat CHRNA4 is expressed in all layers of the frontal cortex (Wevers etal., 1994).

The nAChR is a transmembrane pentamer that is composed of up to fourdifferent subunits (α, β, γ, δ). The nAChRs are found not only in thenervous system but also in skeletal muscle, however in nerve cells, onlytwo types of subunits, α and β, have been identified. Eleven distinctgenes encoding neuronal nAChR subunits (α2-α9 and β2-β4) have been foundin various species to date with the most abundant nAChR subtype inmammalian brain :consisting of two α4 and three β2 (CHRNB2) subunits(Schoepfer et al., 1988; Whiting et al., 1991; Sargent, 1993).

Each subunit of the nAChR consists of a long extracellular domain at theN terminus and four hydrophobic segments (M1-M4) that have sufficientlength to traverse the membrane (reviewed by Jackson, 1999). Thesesubunits associate together into a rosette-like structure with awater-filled pore in the middle. This membrane-spanning pore is lined byfive α-helical segments constituting the M2 domains from each of the 5subunits. These domains, in the absence of the neurotransmitter, ACh,appear to come together near the middle of the membrane and form thegate of the channel. The gate opens upon binding of ACh to distant siteson the α subunits allowing the flow of ions through the channel andcloses again when ACh is depleted from the synaptic cleft or whendesensitization of the receptor occurs. The M2 segment thus is a sitewhere much of the action occurs during channel gating, indicating thatthis domain plays a pivotal role in the process of receptor activation.

Mutation analysis of the CHRNA4 gene in affected family members showinglinkage to this locus identified a missense mutation that replacesserine with phenylalanine at codon 248, a strongly conserved amino acidin the second transmembrane domain (M2) of the encoded protein(Steinlein et al., 1995). This mutation was present in all 21 availableaffected members of the family as well as in 4 obligate carriers and theamino acid change was not seen in 333 healthy control subjects. A secondmutation in this gene was identified in a Norwegian family with the sameform of epilepsy, namely an insertion of three nucleotides at position776, which encodes a leucine (Steinlein et al., 1997). This amino acidinsertion again affected the M2 domain of the CHRNA4 protein.Physiological and pharmacological investigations of human nAChRsreconstituted in Xenopus oocytes with the control or mutated α4 subunitsestablished that both mutations resulted in major but different changesto the receptors. The S248F mutation mainly affected the desensitizationproperties of the receptor while the leucine insertion increased theprobability of transition to the active state (Bertrand et al., 1998).Although these mutations appeared to differentially affect the receptorproperties they both result in reduced permeability to calcium andenhanced desensitization sensitivity that might account for the ADNFLEphenotype.

Genetic linkage studies of additional families with ADNFLE havesuggested that they do not show linkage to the CHRNA4 locus at 20q13.2(Berkovic et al., 1995; Phillips et al., 1998). While one ADNFLE familyshowed linkage to 15q24 (close to the CHRNA3/CHRNA5/CHRNB4 genecluster), in other families both 15q24 and 20q13.2 were excludedindicating that at least three different genes exist accounting forADNFLE. In fact, in at least three other forms of monogenic idiopathicepilepsy, locus heterogeneity has also been demonstrated. These includebenign familial neonatal convulsions (BFNC) mapped to 20q13.2 and8q(Leppert et al., 1989; Lewis et al., 1993), familial febrile seizuresmapped to 8q13-q21and 19p13.3 (Wallace et al., 1996; Johnson et al.,1998) and benign familial infantile convulsions mapped to 19q and 16(Guipponi et al., 1997; Szepetowski et al., 1997). For BFNC, the geneslocated in the 20q13.2 and 8q critical regions that were found to bemutated in individuals with the disease were homologous potassiumchannels (Biervert et al., 1998; Charlier et al., 1998; Singh et al.,1998).

DISCLOSURE OF THE INVENTION

The present inventors have found that the CHRNB2 locus is involved inautosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), and soimplicated the β-subunits of the nAChR in idiopathic epilepsies.

According to one aspect of the present invention there is provided anisolated DNA molecule encoding a mutant β-subunit of a mammaliannicotinic acetylcholine receptor (nAchR), wherein a mutation eventselected from the group consisting of point mutations, deletions,insertions and rearrangements has occurred in the nucleotides encodingthe M2 domain of the β-subunit of said mammalian nicotinic acetylcholinereceptor and said mutation event disrupts the functioning of anassembled mammalian nicotinic acetylcholine receptor comprising theβ-subunit so as to produce an epilepsy phenotype.

Preferably said mutation event is a point mutation. The mutationtypically results in replacement of valine residue. The valine residueis typically replaced by an amino acid having a more bulky side chainand/or a β-carbon atom substituted only by hydrogen atoms, of whichmethionine and leucine are preferred. The valine residue typically formspart of the lining of the ion channel in the vicinity of the opening ofthe channel to the synaptic cleft.

Advantageously said valine is V287 using nomenclature on the NCBIdatabase (V262 in the numbering of Rempel et al (1998)), which occurs asa result of a G to A nucleotide transition at base 1025, as shown in SEQID NO:1. The G to A nucleotide transition creates a NlaIII restrictionenzyme site.

The present invention also encompasses DNA molecules in which one ormore additional mutation events selected from the group consisting ofpoint mutations, deletions, insertions and rearrangements have occurred.Any such DNA molecule will have the mutation associated with epilepsydescribed above and will be functional, but otherwise may varysignificantly from the DNA molecules set forth in SEQ ID NO:1.

The nucleotide sequences of the present invention can be engineeredusing methods accepted in the art for a variety of purposes. Theseinclude, but are not limited to, modification of the cloning,processing, and/or expression of the gene product. PCR reassembly ofgene fragments and the use of synthetic oligonucleotides allow theengineering of the nucleotide sequences of the present invention. Forexample, oligonucleotide-mediated site-directed mutagenesis canintroduce further mutations that create new restriction sites, alterexpression patterns and produce splice variants etc.

As a result of the degeneracy of the genetic code, a number ofpolynucleotide sequences, some that may have minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention includes each and every possiblevariation of a polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequences of the present invention, and all suchvariations are to be considered as being specifically disclosed.

The DNA molecules of this invention include cDNA, genomic DNA, syntheticforms, and mixed polymers, both sense and antisense strands, and may bechemically or biochemically modified, or may contain non-natural orderivatised nucleotide bases as will be appreciated by those skilled inthe art. Such modifications include labels, methylation, intercalators,alkylators and modified linkages. In some instances it may beadvantageous to produce nucleotide sequences possessing a substantiallydifferent codon usage than that of the polynucleotide sequences of thepresent invention. For example, codons may be selected to increase therate of expression of the peptide in a particular prokaryotic oreukaryotic host corresponding with the frequency that particular codonsare utilized by the host. Other reasons to alter the nucleotide sequencewithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringmutated sequence.

The invention also encompasses production of DNA sequences of thepresent invention entirely by synthetic chemistry. Synthetic sequencesmay be inserted into expression vectors and cell systems that containthe necessary elements for transcriptional and translational control ofthe inserted coding sequence in a suitable host. These elements mayinclude regulatory sequences, promoters, 5′ and 3′ untranslated regionsand specific initiation signals (such as an ATG initiation codon andKozak consensus sequence) which allow more efficient translation ofsequences encoding the polypeptides of the present invention. In caseswhere the complete coding sequence, including the initiation codon andupstream regulatory sequences, are inserted into the appropriateexpression vector, additional control signals may not be needed.However, in cases where only coding sequence, or a fragment thereof, isinserted, exogenous translational control signals as described aboveshould be provided by the vector. Such signals may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate for the particularhost cell system used (Scharf et al., 1994).

The invention also includes nucleic acid molecules that are thecomplements of the sequences described herein.

According to another aspect of the present invention there is providedan isolated DNA molecule comprising the nucleotide sequence set forth inSEQ ID NO:1.

According to still another aspect of the present invention there isprovided an isolated DNA molecule consisting of the nucleotide sequenceset forth in SEQ ID NO:1.

The present invention allows for the preparation of purified polypeptideor protein from the polynucleotides of the present invention, orvariants thereof. In order to do this, host cells may be transformedwith a DNA molecule as described above. Typically said host cells aretransfected with an expression vector comprising a DNA moleculeaccording to the invention. A variety of expression vector/host systemsmay be utilized to contain and express sequences encoding polypeptidesof the invention. These include, but are not limited to, microorganismssuch as bacteria transformed with plasmid or cosmid DNA expressionvectors; yeast transformed with yeast expression vectors; insect cellsystems infected with viral expression vectors (e.g., baculovirus); ormouse or other animal or human tissue cell systems. Mammalian cells canalso be used to express a protein using a vaccinia virus expressionsystem. The invention is not limited by the host cell employed.

The polynucleotide sequences, or variants thereof, of the presentinvention can be stably expressed in cell lines to allow long termproduction of recombinant proteins in mammalian systems. Sequencesencoding the polypeptides of the present invention can be transformedinto cell lines using expression vectors which may contain viral originsof replication and/or endogenous expression elements and a selectablemarker gene on the same or on a separate vector. The selectable markerconfers resistance to a selective agent, and its presence allows growthand recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, glycosylation,phosphorylation, and acylation. Post-translational cleavage of a“prepro” form of the protein may also be used to specify proteintargeting, folding, and/or activity. Different host cells havingspecific cellular machinery and characteristic mechanisms forpost-translational activities (e.g., CHO or HeLa cells), are availablefrom the American Type Culture Collection (ATCC) and may be chosen toensure the correct modification and processing of the foreign protein.

When large quantities of the gene are needed, such as for antibodyproduction, vectors which direct high levels of expression of thisprotein may be used, such as those containing the T5 or T7 induciblebacteriophage promoter. The present invention also includes the use ofthe expression systems described above in generating and isolatingfusion proteins which contain important functional domains of theprotein. These fusion proteins are used for binding, structural andfunctional studies as well as for the generation of appropriateantibodies.

In order to express and purify the protein as a fusion protein, theappropriate cDNA sequence is inserted into a vector which contains anucleotide sequence encoding another peptide (for example, glutathioninesuccinyl transferase). The fusion protein is expressed and recoveredfrom prokaryotic or eukaryotic cells. The fusion protein can then bepurified by affinity chromatography based upon the fusion vectorsequence. The desired protein is then obtained by enzymatic cleavage ofthe fusion protein.

Fragments of the polypeptides of the present invention may also beproduced by direct peptide synthesis using solid-phase techniques.Automated synthesis may be achieved by using the ABI 431A PeptideSynthesizer (Perkin-Elmer). Various fragments of this protein may besynthesized separately and then combined to produce the full lengthmolecule.

According to still another aspect of the present invention there isprovided an isolated polypeptide, said polypeptide being a mutantβ-subunit of a mammalian nicotinic acetylcholine receptor (nAChR),wherein a mutation event selected from the group consisting ofsubstitutions, deletions, insertions and rearrangements has occurred inthe M2 domain and said mutation event disrupts the functioning of anassembled mammalian nicotinic acetylcholine receptor so as to produce anepilepsy phenotype.

Typically said mutation event is a substitution involving a valineresidue, which is generally substituted by an amino acid having a morebulky side chain and/or β-carbon atom substituted only by hydrogenatoms. Typically said valine residue is replaced by methionine orleucine.

Preferably the substitution is a V287M transition as illustrated in SEQID NO:2.

The isolated polypeptide of the present invention may have beensubjected to one or more mutation events selected from the groupconsisting of substitutions, deletions, insertions and rearrangements inaddition to the mutation associated with epilepsy. Typically thesemutation events are conservative substitutions.

According to still another aspect of the present invention there isprovided an isolated polypeptide comprising the sequence set forth inSEQ ID NO:2.

According to still another aspect of the present invention there isprovided a polypeptide consisting of the amino acid sequence set forthin SEQ ID NO:2.

According to still another aspect of the present invention there isprovided an isolated polypeptide, said polypeptide being an assembledmammalian nicotinic acetylcholine receptor, comprising at least oneα-subunit and at least one β-subunit, wherein a mutation event selectedfrom the group consisting of substitutions, deletions, insertions andrearrangements has occurred in the M2 domain of a β-subunit and saidmutation event disrupts the functioning of the assembled mammaliannicotinic acetylcholine receptor so as to produce an epilepsy phenotype.

The assembled nicotinic acetylcholine receptor may contain mutations ina single β-subunit or in a number of β-subunits. The main functionalnAchR in the brain is a pentameric molecule consisting of two α4subunits and three β2 subunits, and a mutation event may have occurredin any or all of the β2 subunits.

According to still another aspect of the present invention there isprovided a method of preparing a polypeptide, said polypeptide being amutant β-subunit of a mammalian nicotinic acetylcholine receptor,comprising the steps of:

(1) culturing host cells transfected with an expression vectorcomprising a DNA molecule as described above under conditions effectivefor polypeptide production; and

(2) harvesting the mutant β-subunit.

The mutant β-subunit may also be allowed to assemble with wild-typeβ-subunits and other subunits of the nicotinic acetylcholine receptor,whereby the assembled nAChR is harvested.

Substantially purified protein or fragments thereof can then be used infurther biochemical analyses to establish secondary and tertiarystructure for example by X-ray crystallography of crystals of theproteins or by NMR. Determination of structure allows for the rationaldesign of pharmaceuticals to interact with the nAChR through a specificsubunit protein, alter the overall nAChR protein charge configuration orcharge interaction with other proteins, or to alter its function in thecell.

It will be appreciated that, having identified a mutation involved inepilepsy (ADNFLE) in these proteins, the nAChRs will be useful infurther applications which include a variety of hybridisation andimmunological assays to screen for and detect the presence of either anormal or mutated gene or gene product.

The invention enables therapeutic methods for the treatment of epilepsy,particularly ADNFLE, and also enables methods for the diagnosis ofidiopathic epilepsies.

According to still another aspect of the invention there is provided amethod of treating epilepsy, comprising administering a selectiveantagonist of the nicotinic acetylcholine receptor when it contains amutation in the M2 domain of a β-subunit, said mutation being causativeof epilepsy, to a subject in need of such treatment.

In still another aspect of the invention there is provided the use of aselective antagonist of the nAChR when it contains a mutation in the M2domain of β-subunit, said mutation being causative of epilepsy, in themanufacture of a medicament for the treatment of epilepsy.

In one aspect, an antibody, which specifically binds to a mutant nAChR,may be used directly as an antagonist or indirectly as a targeting ordelivery mechanism for bringing a pharmaceutical agent to cells ortissues that express the nAChR.

In a still further aspect of the invention there is provided an antibodywhich is immunologically reactive with a polypeptide as described above,but not with a wild-type nicotinic acetylcholine receptor or subunitthereof.

In particular, there is provided an antibody to an assembled nAChRcontaining a mutation causative of epilepsy in the M2 domain of aβ-subunit. The antibody may be a monoclonal antibody or polyclonalantibody as would be understood by the person skilled in the art.

Alternatively, in some mutants, it may be possible to prevent thedisorder by introducing another copy of the homologous subunit genebearing a second mutation in that gene, or to alter the mutation, or touse another gene to block any negative effect.

In a further aspect of the invention there is provided a method oftreating epilepsy, comprising administering an isolated DNA moleculewhich is the complement of any one of the DNA molecules described aboveand which encodes a mRNA that hybridizes with the mRNA encoding themutant β-subunits of the nAChR, to a subject in need of such treatment.

In a still further aspect of the invention there is provided the use ofan isolated DNA molecule which is the complement of a DNA molecule ofthe invention and which encodes a mRNA that hybridizes with the mRNAencoding the mutant β-subunits of the nAChR, in the manufacture of amedicament for the treatment of epilepsy.

Typically, a vector expressing the complement of the polynucleotideencoding the subunits constituting the nAChR may be administered to asubject to treat or prevent epilepsy, particularly ADNFLE. Antisensestrategies may use a variety of approaches including the use ofantisense oligonucleotides, injection of antisense RNA and transfectionof antisense RNA expression vectors. Many methods for introducingvectors into cells or tissues are available and equally suitable for usein vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may beintroduced into stem cells taken from the patient and clonallypropagated for autologous transplant back into that same patient.Delivery by transfection, by liposome injections, or by polycationicamino polymers may be achieved using methods which are well known in theart. (For example, see Goldman et al., 1997).

In further embodiments, any of the antagonists, antibodies,complementary sequences or vectors of the invention may be administeredin combination with other appropriate therapeutic agents. Selection ofthe appropriate agents may be made by those skilled in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, therapeutic efficacy with lower dosages of each agent may bepossible, thus reducing the potential for adverse side effects.

Using methods well known in the art, a selective antagonist of a mutantnAChR may be produced. In particular, a mutant nAChR may be used toproduce antibodies specific for the mutant β-subunits causative of theidiopathic epilepsies or to screen libraries of pharmaceutical agents toidentify those that specifically bind the mutant nAChR. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies.

For the production of antibodies, various hosts including rabbits, rats,goats, mice, humans, and others may be immunized by injection with apolypeptide as described or with any fragment or oligopeptide thereof(provided it includes the mutation of the invention) which hasimmunogenic properties. Various adjuvants may be used to increaseimmunological response and include, but are not limited to, Freund's,mineral gels such as aluminum hydroxide, and surface-active substancessuch as lysolecithin. Adjuvants used in humans include BCG (bacilliCalmette-Guerin) and Corynebacterium parvum.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to the mutant nAChR have an amino acid sequenceconsisting of at least about 5 amino acids, and, more preferably, of atleast about 10 amino acids. It is also preferable that theseoligopeptides, peptides, or fragments are identical to a portion of theamino acid sequence of the natural protein and contain the entire aminoacid sequence of a small, naturally occurring molecule. Short stretchesof nAChR amino acids may be fused with those of another protein, such asKLH, and antibodies to the chimeric molecule may be produced.

Monoclonal antibodies to a mutant nAChR may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique. (For example, see Kohler et al., 1975; Kozboret al., 1985; Cote et al., 1983; Cole et al., 1984).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature. (Forexample, see Orlandi et al., 1989; Winter et al., 1991).

Antibody fragments which contain specific binding sites for a nAChR mayalso be generated. For example, such fragments include, F(ab′)2fragments produced by pepsin digestion of the antibody molecule and Fabfragments generated by reducing the disulfide bridges of the F(ab′)2fragments. Alternatively, Fab expression libraries may be constructed toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity. (For example, see Huse et al., 1989).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between a nAChR and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering nAChR epitopes is preferred, but a competitivebinding assay may also be employed.

According to still another aspect of the invention, peptides of theinvention, particularly purified mutant nAChR polypeptide and cellsexpressing these, are useful for the screening of candidatepharmaceutical agents in a variety of techniques. It will be appreciatedthat therapeutic agents useful in the treatment of the idiopathicepilepsies such as ADNFLE are likely to show binding affinity to thepolypeptides of the invention. Such techniques include, but are notlimited to, high-throughput screening for compounds having suitablebinding affinity to the mutant nAChR polypeptides (see PCT publishedapplication WO84/03564). In this stated technique, large numbers ofsmall peptide test compounds can be synthesised on a solid substrate andcan be assayed through nAChR polypeptide binding and washing. BoundnAChR polypeptide is then detected by methods well known in the art. Ina variation of this technique, purified polypeptides of the inventioncan be coated directly onto plates to identify interacting testcompounds. The invention also contemplates the use of competition drugscreening assays in which neutralizing antibodies capable ofspecifically binding the mutant nAChR compete with a test compound forbinding thereto. In this manner, the antibodies can be used to detectthe presence of any peptide that shares one or more antigenicdeterminants of the mutant nAChR.

The invention is particularly useful for screening compounds by usingthe polypeptides of the invention in transformed cells, transfectedoocytes or transgenic animals. A particular drug is added to the cellsin culture or administered to a transgenic animal containing mutantnAChRs and the effect on the current of the receptor is compared to thecurrent of a cell or animal containing the wild-type nAChR. Drugcandidates that alter the current to a more normal level are useful fortreating or preventing diseases associated with nAChRs.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

Polynucleotide sequences encoding a nAChR may be used for the diagnosisof the idiopathic epilepsies such as ADNFLE and the use of the DNAmolecules of the invention in diagnosis of epilepsy, or a predispositionto epilepsy, is therefore contemplated.

In another embodiment of the invention, the polynucleotides that may beused for diagnostic purposes include oligonucleotide sequences, genomicDNA and complementary RNA and DNA molecules. The polynucleotides may beused to detect and quantitate gene expression in biological samples.Genomic DNA used for the diagnosis may be obtained from body cells, suchas those present in the blood, tissue biopsy, surgical specimen, orautopsy material. The DNA may be isolated and used directly fordetection of a specific sequence or may be amplified by the polymerasechain reaction (PCR) prior to analysis. Similarly, RNA or cDNA may alsobe used, with or without PCR amplification. To detect a specific nucleicacid sequence, hybridization using specific oligonucleotides, PCRmapping, RNase protection, and various other methods may be employed.For instance restriction enzyme digest and mapping can be employed forthe specific G to A mutation in the CHRNB2 subunit described in thisinvention. The G to A transition in the M2 domain of this subunitcreates a NlaIII restriction site. The DNA from an affected individualas well as a normal control may be amplified using oligonucleotidesdescribed in SEQ ID NO: 3 and 4. The amplification product may then bedigested by NlaIII to provide a fingerprint for comparison to the DNAfingerprint of wild-type CHRNB2. In addition, direct nucleotidesequencing of amplification products from the nAChR can be employed.Sequence of the sample amplicon is compared to that of the wild-typeamplicon to determine the presence (or absence) of nucleotidedifferences.

According to a further aspect of the invention there is provided the useof a polypeptide as described above in the diagnosis of epilepsy.

When a diagnostic assay is to be based upon proteins constituting anAChR, a variety of approaches are possible. For example, diagnosis canbe achieved by monitoring differences in the electrophoretic mobility ofnormal and mutant proteins that form the nAChR. Such an approach will beparticularly useful in identifying mutants in which charge substitutionsare present, or in which insertions, deletions or substitutions haveresulted in a significant change in the electrophoretic migration of theresultant protein. Alternatively, diagnosis may be based upondifferences in the proteolytic cleavage patterns of normal and mutantproteins, differences in molar ratios of the various amino acidresidues, or by functional assays demonstrating altered function of thegene products.

In another aspect, antibodies that specifically bind mutant nAChRs maybe used for the diagnosis of epilepsy, or in assays to monitor patientsbeing treated with a complete nAChR or agonists, antagonists, orinhibitors of a nAChR. Antibodies useful for diagnostic purposes may beprepared in the same manner as described above for therapeutics.Diagnostic assays for nAChRs include methods that utilize the antibodyand a label to detect a mutant nAChR in human body fluids or in extractsof cells or tissues. The antibodies may be used with or withoutmodification, and may be labelled by covalent or non-covalent attachmentof a reporter molecule.

A variety of protocols for measuring the presence of mutant nAChRs,including ELISAs, RIAs, and FACS, are known in the art and provide abasis for diagnosing epilepsies such as ADNFLE. The expression of amutant receptor is established by combining body fluids or cell extractstaken from test mammalian subjects, preferably human, with antibody tothe receptor under conditions suitable for complex formation. The amountof complex formation may be quantitated by various methods, preferablyby photometric means. Antibodies specific for the mutant receptor willonly bind to individuals expressing the said mutant receptor and not toindividuals expressing only wild-type receptors (ie normal individuals).This establishes the basis for diagnosing the disease.

Once an individual has been diagnosed with epilepsy, effectivetreatments can be initiated. These may include administering a selectiveantagonist to the mutant receptor such as an antibody or mutantcomplement as described above. This therapy can also be supported withthe introduction of wild-type receptor, particularly through genetherapy approaches. Typically, a vector capable of a expressing theappropriate full length nAChR subunit or a fragment of derivativethereof may be administered. The expression vector must be able to driveits own expression such that the level of normal protein will besufficient for normal receptor formation.

In an alternative support approach to therapy, a substantially purifiednAChR or nAChR subunit polypeptide and a pharmaceutically acceptablecarrier may be administered. Pharmaceutical compositions in accordancewith the present invention are prepared by mixing nAChR or nAChR subunitpolypeptide or active fragments or variants thereof having the desireddegree of purity, with acceptable carriers, excipients, or stabilizerswhich are well known. Acceptable carriers, excipients or stabilizers arenontoxic at the dosages and concentrations employed, and include bufferssuch as phosphate, citrate, and other organic acids; antioxidantsincluding absorbic acid; low molecular weight (less than about 10residues) polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitrol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween, Pluronics orpolyethylene glycol (PEG).

Any of the proteins, antagonists, antibodies, agonists, complementarysequences, or vectors of the invention may be administered incombination with other appropriate therapeutic agents. Selection of theappropriate agents may be made by those skilled in the art, according toconventional pharmaceutical principles. The combination of therapeuticagents may act synergistically to effect the treatment or prevention ofepilepsy. Using this approach, therapeutic efficacy with lower dosagesof each agent may be possible, thus reducing the potential for adverseside effects.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents. Microarrays may beprepared, used, and analyzed using methods known in the art. (Forexample, see Schena et al., 1996; Heller et al., 1997).

The present invention also provides for the production of geneticallymodified (knock-out or knock-in), non-human animal models transformedwith the DNA molecules of the invention. These animals are useful forthe study of the function of a nAChR, to study the mechanisms of diseaseas related to a nAChR, for the screening of candidate pharmaceuticalcompounds, for the creation of explanted mammalian cell cultures whichexpress the mutant nAChR and for the evaluation of potential therapeuticinterventions.

Animal species which are suitable for use in the animal models of thepresent invention include, but are not limited to, rats, mice, hamsters,guinea pigs, rabbits, dogs, cats, goats, sheep, pigs, and non-humanprimates such as monkeys and chimpanzees. For initial studies,genetically modified mice and rats are highly desirable due to theirrelative ease of maintenance and shorter life spans. For certainstudies, transgenic yeast or invertebrates may be suitable and preferredbecause they allow for rapid screening and provide for much easierhandling. For longer term studies, non-human primates may be desired dueto their similarity with humans.

To create an animal model for a mutated nAChR several methods can beemployed. These include generation of a specific mutation in ahomologous animal gene, insertion of a wild type human gene and/or ahumanized animal gene by homologous recombination, insertion of a mutant(single or multiple) human gene as genomic or minigene cDNA constructsusing wild type or mutant or artificial promoter elements or insertionof artificially modified fragments of the endogenous gene by homologousrecombination. The modifications include insertion of mutant stopcodons, the deletion of DNA sequences, or the inclusion of recombinationelements (lox p sites) recognized by enzymes such as Cre recombinase.

To create a transgenic mouse, which is preferred, a mutant version of aparticular nAChR subunit can be inserted into a mouse germ line usingstandard techniques of oocyte microinjection or transfection ormicroinjection into embryonic stem cells. Alternatively, if it isdesired to inactivate or replace an endogenous nAChR subunit gene,homologous recombination using embryonic stem cells may be applied.

For oocyte injection, one or more copies of the mutant nAChR subunitgene can be inserted into the pronucleus of a just-fertilized mouseoocyte. This oocyte is then reimplanted into a pseudo-pregnant fostermother. The liveborn mice can then be screened for integrants usinganalysis of tail DNA for the presence of the particular human subunitgene sequence. The transgene can be either a complete genomic sequenceinjected as a YAC, BAC, PAC or other chromosome DNA fragment, a cDNAwith either the natural promoter or a heterologous promoter, or aminigene containing all of the coding region and other elements found tobe necessary for optimum expression.

According to still another aspect of the invention there is provided theuse of genetically modified non-human animals for the screening ofcandidate pharmaceutical compounds.

It will be clearly understood that, although a number of prior artpublications are referred to herein, this reference does not constitutean admission that any of these documents forms part of the commongeneral knowledge in the art, in Australia or in any other country.

Throughout this specification and the claims, the words “comprise”,“comprises” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred forms of the invention are described, by way of example only,with reference to the following examples and the accompanying drawings,in which:

FIG. 1 is a chart of the lineage of a Scottish family showing the familymembers with the mutated CHRNB2 amino acid 287 (indicated by m);

FIG. 2 is a trace of the DNA sequence of CHRNB2 showing the c1025G-Atransition in which the upper chromatogram shows the mutation and thelower chromatogram shows the control sequence;

FIG. 3 shows the alignment of various genes in order to allow acomparison of the homologies at the M2 CHRNB2 domain in which amino acid287 is indicated by the box; and

FIG. 4 shows A, left panel, Neuronal α4β2 heteropentameric receptorresulting from the assembly of two α4 and three β2 subunits. Rightpanel, α4β2 is shown as a pentamer with two potential ACh-blindingsites. “β2*” identifies the V287M amino acid substitution. The AChreceptor can assemble without any mutated β2 subunit (α4β2 wild-typereceptor) or with one, two, or three mutated β2* subunits. B, upperpanel, Current traces elicited by consecutive applications of four orfive increasing ACh concentrations (horizontal bars). The values aboveeach bar indicate the concentration of ACh applied to the cell. Lowerpanel, Differences in ACh affinity are emphasized in a log-log plot. Thelogarithm of the absolute value of the current (measured in the upperpanel, Log (−I) is plotted as a function of the logarithm of AChconcentrations (Log [ACh]). −I and [ACh] are expressed in nA and nM,respectively. Values are mean ±SEM, with n=9 (α4β2), n=17 (α4β2*), andn=10 (α4[β2*+β2]). Solid lines are the best linear regression throughoutdata points. C, upper panel, Representative macroscopic currentsrecorded in oocytes expressing either the wild-type or mutated β2subunit. Currents evoked by four ACh concentrations (0.1, 0.2, 0.8 and 8μM) are superimposed. Cells were held at −100 mV and challenged with ACh(10 s) once every 120 s. Bars indicate ACh applications. Lower panel,ACh activation curves for α4β2, α4β2* and α4(β2*+β2]). Dose-responsecurves were normalized to the maximal current amplitude of each cell.Values are mean ±SEM. For each ACh concentration, 3-8 independentmeasurements were averaged.

MODES FOR PERFORMING THE INVENTION EXAMPLE 1 Clinical Diagnosis ofAffected Family Members

The proband (V-1; FIG. 1), a Caucasian female of normal intellect,presented at the age of 11 with nocturnal seizures. These would betypified by her waking from sleep with a sensation of difficulty inbreathing. This would last a few seconds only, following which she wouldappear to be holding her breath and would make grunting noises.Sometimes she would recover quickly which would be followed by crying orscreaming. On other occasions this would progress to tonic extension ofher left arm and curling up into a ball. This would last up to a fewminutes and the whole episode would be repeated stereotypically ataround 15 minute intervals throughout the night. She would have a clearrecollection of all of the above events. Such events would also occurduring daytime sleep.

Video EEG telemetry showed no change during the ictus, although the EEGtrace was largely obscured by muscle artifact. Inter-ictal recordingshave on occasion shown some sharp disturbance in the right centralparietal area.

Whilst her seizures were initially well controlled using carbamazepine,this relapsed and further control was only achieved with somedifficulty. Currently, she is seizure free on a combination of phenytoinand topiramate.

In this family there are nine other affected members spanning fourgenerations. The symptoms in these individuals have either been so mildthat medical attention has not been sought or their symptoms have beeneasily controlled on carbamazepine. In some family members the seizureshave remitted spontaneously but the oldest surviving family member(II-2; FIG. 1), at the age of 81, continues to have occasional seizureswhilst on phenytoin.

EXAMPLE 2 Mutation Analysis of CHRNB2

The second transmembrane domains of the CHRNB2 gene were screened bydirect sequencing of DNA obtained with consent from eight members of thefamily: six affected individuals, one obligate carrier and hisunaffected wife (FIG. 1). DNA was extracted from peripheral bloodsamples using a method adapted from Wyman and White (1980). CHRNB2specific primers used to amplify the extracted DNA are listed as SEQ IDNO: 3 and 4. These primers amplify a 468 base pair segment of the CHRNB2gene that incorporates the M2 domain. PCR reactions contained 67 mMTris-HCl (pH 8.8); 16.5 mM (NH₄)₂SO₄; 6.5 μM EDTA; 1.5 mM MgCl₂; 200 μMeach DNTP; 10% DMSO; 0.17 mg/ml BSA; 10 mM β-mercaptoethanol; 15 μg/mleach primer; 100 U/ml Taq DNA polymerase, and 10 μg/ml genomic DNA. PCRreactions were performed using 10 cycles of 94° C. for 60 seconds, 60°C. for 90 seconds, and 72° C. for 90 seconds followed by 25 cycles of94° C. for 60 seconds, 55° C. for 90 seconds, and 72° C. for 90 seconds.A final extension reaction for 10 minutes at 72° C. followed.

PCR amplified templates were purified for sequencing using QiaQuick PCRpreps (Qiagen) following manufacturers procedures. The primers used tosequence the purified CHRNB2 PCR fragments were identical to those usedfor the initial amplification step (SEQ ID Numbers: 3 and 4). For eachsequencing reaction, 25 ng of primer and 100 ng of purified PCR templatewere used. The BigDye sequencing kit (ABI) was used for all sequencingreactions according to the manufacturers specifications. The productswere run on an ABI 377 Sequencer and analysed using the EditViewprogram.

The sequencing strategy revealed a G→A transition in the M2 domain ofCHRNB2 in the proband and in other family members where the presence ofthe mutation has been indicated (FIG. 1). The nucleotide sequence of themutated form of the CHRNB2 gene is represented by SEQ ID NO: 1. Thec1025G→A mutation (FIG. 2) replaces a highly conserved valine with amethionine at position 287, using nomenclature on the NCBI database, orat position 262, using the numbering of Rempel et al., (1998). The aminoacid sequence of the mutated form of the CHRNB2 gene is represented bySEQ ID NO: 2. All affected individuals and the unaffected obligatecarrier had the mutation There is much interspecies and between subunithomology among CHRN subunits, especially in the four transmembranedomains. The CHRNB subunits CHRNB2, CHRNB3 and CHRNB4 are all expressedin the brain. The M2 domain of CHRNB3 has only 59% homology with theother two and in vitro studies in the rat show that, when coexpressedwith CHRNA subunits alone, CNRNB3 does not assemble into a functionalreceptor. The β-type subunits therefore may function in ion channelsgated by ligands other than nicotine and acetylcholine (Willoughby etal., 1993). The M2 domains of CHRNB2 and CHRNB4 have almost completehomology and in particular valine287 is fully conserved in thesesubunits as well as in a number of other species (FIG. 3). CHRNB1, whichis expressed only in muscle, has leucine at this position. This mayindicate valine 287 is essential for normal ion channel function in thebrain. The V287M mutation is located near the extracellular end of theM2 domain that lines the ionic pore (FIG. 4A). Valine 287 faces into thepore of the ion channel in the open and closed state (Devilliers-Thieryet al., 1993) and when valine 287 is replaced by a methionine there isan apparent 10-fold increase in Ach affinity.

EXAMPLE 3 Confirmation of the V287M Mutation—Restriction Enzyme Analysis

The G→A transition creates a NlaIII restriction site. Primersrepresented by SEQ ID Numbers: 3 and 4 amplify a 468 base pair fragmentthat contains four NlaIII sites present in normal alleles. Digestion ofthis normal amplicon with NlaIII will produce fragments of 318, 78, 54,9 and 9 base pairs. However, digestion of an amplicon containing the G→Atransition with NlaIII will produce fragments of 273, 78, 54, 45, 9 and9 base pairs. The bands of 318 and 273 base pairs are easily detected on2% agarose gels and therefore this system provides a mechanism toconfirm the presence of the mutation in affected family members.

EXAMPLE 4 Confirmation of the V287M Mutation—SSCP Analysis

To confirm that the observed amino acid substitution found in the CHRNB2M2 domain was specific to affected members of the studied family, singlestrand conformation polymorphism analysis was performed on DNA collectedfrom 102 anonymous Australian blood donors. The primers used for PCRamplification of DNA from both control donor samples and affected familymembers are represented by SEQ ID Numbers: 4 and 5. These primersamplified a product of 220 base pairs using the following conditions:PCR reactions contained 67 mM Tris-HCl (pH 8.8); 16.5 mM (NH₄)₂SO₄; 6.5μM EDTA; 1.5 mM MgCl₂; 200 μA each dNTP; 10% DMSO; 0.17 mg/ml BSA; 10 mMβ-mercaptoethanol; 15 μg/ml each primer; 200 μCi/ml [α-32P]dCTP; 100U/ml Taq DNA polymerase, and 10 μg/ml genomic DNA. PCR reactions wereperformed using 10 cycles of 94° C. for 60 seconds, 60° C. for 90seconds, and 72° C. for 90 seconds followed by 25 cycles of 94° C. for60 seconds, 55° C. for 90 seconds, and 72° C. for 90 seconds. A finalextension reaction for 10 minutes at 72° C. followed. Completed PCRreactions were subsequently mixed with an equal volume of formamideloading buffer (96% formamide; 1 mM EDTA; 0.1% bromophenol blue; 0.1%xylene cyanol) and were heated to 95° C. for 3 minutes before snapcooling on ice. Five μl of each sample was then loaded onto gelscontaining 10% (49:1) polyacrylamide, 5% glycerol and TBE. The gels wererun at 700 volts for 20 hours at room temperature, dried and exposed toX-ray film.

A bandshift was identified, however the shift was only observed infamily members with the G→A transition and not in any of the 102 controlDNA samples. The bandshift detected by SSCP analysis could only beassociated with the G→A transition, since no other base changes weredetected in the PCR product. The fact that the nucleotide change onlyoccurs in affected family members and not in healthy controls suggeststhe change has a functional significance.

EXAMPLE 5 Functional Significance of the V287M Mutation

To define the effects of the β2 V287M mutation on the physiologicalproperties of the α4β2 nAChR, the V287M amino acid substitution wasintroduced into the β2 coding sequence according to the PCR basedstrategy described by Nelson and Long (1989). The 411 base pairNheI/PmlI mutagenesis cassette was sequenced to verify the presence ofthe V287M mutation. Xenopus laevis oocytes at stage V or VI wereisolated and nuclear-injected with 2 ng of DNA solution based on astandard procedure (Bertrand et al., 1991). For functionalreconstitution of α4β2, α4β2V287M and α4(β2+β2V287M) receptors, cDNAscoding for CHRNA4 (Monteggia et al., 1995), CHRNB2 and CHRNB2-V287Msubunits were mixed at molecular ratios of 1:1, 1:1 and 2:1:1respectively. Following DNA injection, oocytes were maintained at 18° C.for 2 to 3 days in standard Barth's solution containing 88 mM NaCl, 1 mMKCl, 2.4 mM NaHCO₃, 0.8 mM MgSO₄, 0.3 mM Ca(NO₃)₂, 0.4 mM CaCl₂, 10 mMHEPES-NaOH, pH 7.4 and were complemented with kanamycin (20 μg/ml),penicillin (100 μg/ml) and streptomycin (100 μg/ml).

Macroscopic currents were recorded by a two-electrode voltage clamp at18° C. using a GENECLAMP 500 amplifier (Axon Instruments). Theborosilicate electrodes were filled with 3 M KCl and the oocytes werecontinuously perfused a solution containing 82.5 mM NaCl, 2.5 mM KCl,2.5 mM CaCl₂, 1 mM MgCl₂, 0.5 μM atropine sulphate, 5 mM HEPES-NaOH, pH7.4. Ach (Fluka) was diluted in this solution for the subsequentexperiments. Gravity driven solutions were applied to the recordingchamber through computer controlled electromagnetic valves. For allexperiments, the holding potential was −100 mV.

When challenged with saturating concentrations of Ach, the α4β2 andα4β2V287M containing receptors yielded robust currents (>5 μA). However,at low concentrations of Ach, distinction between these two types ofreceptors was observed. With oocytes containing the α4β2V287M subunits,3 nM Ach was already sufficient to activate reliable ionic currents(81±24 nA, n=18) compared with wild-type receptors which needed 50 nMAch to evoke currents of similar amplitude (65±22 nA, n=9)(FIG. 4B).

To characterize these differences further, currents evoked by four orfive consecutive applications of increasing non- orslightly-desensitizing Ach concentrations was recorded. Representativecurrent recordings are shown in the upper panel of FIG. 4B. The lowerpanel of FIG. 4B shows a plot of the logarithm of Ach-evoked current(absolute value) versus the logarithm of agonist concentrations whichindicated the expected linear relationship. The α4β2V287M exhibited ˜1order of magnitude higher Ach sensitivity than did the wild-typereceptors which confirmed the initial qualitative observation thatmutated receptors are more sensitive to Ach.

To further examine Ach sensitivity, the dose-response relationship overa broad range of concentrations was determined for both control andmutated receptors. FIG. 4C (upper panel) shows superimposed currentsevoked by 0.1, 0.2, 0.8 and 8 μM external Ach. The Ach sensitivity wasdetermined by plotting peak current versus the logarithm of agonistconcentration (FIG. 4C, lower panel). Observation of the α4β2 activationcurves suggest that they are best described by the sum of two Hillequations (See below, solid lines in FIG. 4C, lower panel). Most of theAch activation curves appeared biphasic with high- and low-affinitycomponents and consequently Ach dose-response relationships are bestfitted to the sum of two empirical Hill equations:I=I _(max) {[a/(1+EC50H/[ACh])^(nH)]+(1−a)/(1+EC50L/[ACh] ^(nL))}where I_(maw) is the amplitude of the maximal current elicited by AChapplication, EC50 is the ACh concentration for half-maximal currentactivation, [ACh] is the concentration of ACh, and n is the Hillcoefficient. Parameters relating to the high- and low-affinity componentare identified by H and L respectively. Parameter a is the relativecontribution of the high-affinity component to the total currentresponse over the range of concentrations and is expressed as thefraction of the high-affinity sites. Table 1 shows the values for theseparameters. Within the limits of the model used for the data fit, theα4β2V287M receptor exhibited both a decrease in the EC50s and anincrease in the relative contribution of the high-affinity component.Thus, both log-log plot and dose-response curves accounted for a higherapparent ACh affinity associated with the β2V287M mutation.

The peak versus plateau current ratio revealed no major differencesbetween the wild-type and mutated receptors indicating that the V287Mmutation causes no significant alteration of desensitization properties.

As the affected family members of the present study are heterozygous forthe β2 gene, all cells from these individuals most likely express bothwild-type and mutated subunits. Therefore experiments that examine theeffect of co-injection of both wild-type and mutated β2 subunits in thesame oocyte are essential. Results showed that within the same batch ofoocytes, currents evoked by saturating ACh concentrations wereindistinguishable on the basis of their amplitude. In contrast, anobvious difference was observed for the apparent ACh affinity. Both thehigh and low EC50 values were comparable to receptors containing threeV287M β2 subunits (Table 1). In addition, α4(β2+β2V287M), like α4β2V287Mexhibited a higher affinity in the log-log plot (FIG. 4B).

These findings are consistent with the autosomal dominant mode ofinheritance of ADNFLE. FIG. 4A indicates that four distinct subunitcombinations are possible when both wild-type and mutated subunits areexpressed in a cell. Based on this, 75% of the receptors will contain amutated β2 subunit and the dominant effect of the mutation can thereforebe easily accounted for. As oocytes that express heterozygote orhomozygote combinations of the mutated β2 subunit display very similarproperties, the presence of a single β2 mutation within a receptorcomplex may be sufficient to confer the properties associated with theV287M substitution.

EXAMPLE 6 Analysis of the nAChR and Receptor Subunits

The following methods are used to determine the structure and functionof the nAChR and receptor subunits.

Molecular Biological Studies

The ability of nAChR as a whole or through individual subunits to bindknown and unknown protein can be examined. Procedures such as the yeasttwo-hybrid system are used to discover and identify any functionalpartners. The principle behind the yeast two-hybrid procedure is thatmany eukaryotic transcriptional activators, including those in yeast,consist of two discrete modular domains. The first is a DNA-bindingdomain that binds to a specific promoter sequence and the second is anactivation domain that directs the RNA polymerase II complex totranscribe the gene downstream of the DNA binding site. Both domains arerequired for transcriptional activation as neither domain can activatetranscription on its own. In the yeast two-hybrid procedure, the gene ofinterest or parts thereof (BAIT), is cloned in such a way that it isexpressed as a fusion to a peptide that has a DNA binding domain. Asecond gene, or number of genes, such as those from a cDNA library(TARGET), is cloned so that it is expressed as a fusion to an activationdomain. Interaction of the protein of interest with its binding partnerbrings the DNA-binding peptide together with the activation domain andinitiates transcription of the reporter genes. The first reporter genewill select for yeast cells that contain interacting proteins (thisreporter is usually a nutritional gene required for growth on selectivemedia). The second reporter is used for confirmation and while beingexpressed in response to interacting proteins it is usually not requiredfor growth.

The nature of the nAChR interacting genes and proteins can also bestudied such that these partners can also be targets for drug discovery.

Structural Studies

nAChR recombinant proteins can be produced in bacterial, yeast, insectand/or mammalian cells and used in crystallographical and NMR studies.Together with molecular modelling of the protein, structure-driven drugdesign can be facilitated.

EXAMPLE 7 Generation of Polyclonal Antibodies Against nAChR Subunits

Following the identification of a mutation in the β2 subunit of thenAChR in ADNFLE and therefore confirming the involvement of the receptorin epilepsy, antibodies can be made to selectively bind and distinguishmutant from normal protein. Antibodies specific for mutagenised epitopesare especially useful in cell culture assays to screen for cells whichhave been treated with pharmaceutical agents to evaluate the therapeuticpotential of the agent.

To prepare polyclonal antibodies, short peptides can be designedhomologous to a particular nAChR subunit amino acid sequence. Suchpeptides are typically 10 to 15 amino acids in length. These peptidesshould be designed in regions of least homology to the mouse orthologueto avoid cross species interactions in further down-stream experimentssuch as monoclonal antibody production. Synthetic peptides can then beconjugated to biotin (Sulfo-NHS-LC Biotin) using standard protocolssupplied with commercially available kits such as the PIERCE™ kit(PIERCE). Biotinylated peptides are subsequently complexed with avidinin solution and for each peptide complex, 2 rabbits are immunized with 4doses of antigen (200 μg per dose) in intervals of three weeks betweendoses. The initial dose is mixed with Freund's Complete adjuvant whilesubsequent doses are combined with Freund's Immuno-adjuvant. Aftercompletion of the immunization, rabbits are test bled and reactivity ofsera assayed by dot blot with serial dilutions of the original peptides.If rabbits show significant reactivity compared with pre-immune sera,they are then sacrificed and the blood collected such that immune seracan separated for further experiments.

This procedure is repeated to generate antibodies against wild-typeforms of receptor subunits. These antibodies, in conjunction withantibodies to mutant nAChR subunits, are used to detect the presence andthe relative level of the mutant forms in various tissues.

EXAMPLE 8 Generation of Monoclonal Antibodies Specific for nAChRSubunits

Monoclonal antibodies can be prepared for nAChR subunits in thefollowing manner. Immunogen comprising an intact nAChR subunit proteinor nAChR subunit peptides (wild type or mutant) is injected in Freund'sadjuvant into mice with each mouse receiving four injections of 10 to100 ug of immunogen. After the fourth injection blood samples taken fromthe mice are examined for the presence of antibody to the immunogen.Immune mice are sacrificed, their spleens removed and single cellsuspensions are prepared (Harlow and Lane, 1988). The spleen cells serveas a source of lymphocytes, which are then fused with a permanentlygrowing myeloma partner cell (Kohler and Milstein, 1975). Cells areplated at a density of 2×10⁵ cells/well in 96 well plates and individualwells are examined for growth. These wells are then tested for thepresence of nAChR subunit specific antibodies by ELISA or RIA using wildtype or mutant subunit target protein. Cells in positive wells areexpanded and subcloned to establish and confirm monoclonality. Cloneswith the desired specificity are expanded and grown as ascites in micefollowed by purification using affinity chromatography using Protein ASepharose, ion-exchange chromatography or variations and combinations ofthese techniques.

Industrial Applicability

The present invention allows for the diagnosis and treatment ofidiopathic epilepsies such as ADNFLE. TABLE 1 Properties of Wild-typeand β2-V287M Containing Receptors Subunit Combinations EC50 (H) EC50 (L)n (H) n (L) % (H) % (L) Number α4β2 3.9 ± 1.3 47.6 ± 1.88 1.2 ± 0.2 1.3± 0.2 26 ± 5 74 ± 5 8 α4β2* 0.25 ± 0.04 2.9 ± 0.9 1.6 ± 0.1 1.9 ± 0.1 77± 1.5 23 ± 1.5 7 α4 (β2* + β2) 0.42 ± 0.14 5.3 ± 2.3 1.6 ± 0.1 1.0 ± 0.165 ± 3 35 ± 3 7Note:Sensitivity to ACh was measured in several cells (n >= 5). Valuescorrespond to the best fits obtained with Equation 1.Values are mean ± SEM.*Subunits with the V287M mutation.Equation 1:I = I_(max){[a/(1 + EC50H/[ACh])^(nH)] + (1 − a)/(1 + EC50L/[ACh]^(nL))}References

References cited herein are listed on the following pages, and areincorporated herein by this reference.

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1. An isolated DNA molecule encoding a mutant β-subunit of a mammaliannicotinic acetylcholine receptor (nAchR), wherein a mutation eventselected from the group consisting of point mutations, deletions,insertions and rearrangements has occurred in the nucleotides encodingthe M2 domain of the β-subunit of said mammalian nicotinic acetylcholinereceptor and said mutation event disrupts the functioning of anassembled mammalian nicotinic acetylcholine receptor comprising theβ-subunit so as to produce an epilepsy phenotype.
 2. An isolated DNAmolecule as claimed in claim 1 wherein said mutation event is a pointmutation.
 3. An isolated:DNA molecule as claimed in claim 2 wherein saidpoint mutation results in substitution of a valine residue.
 4. Anisolated DNA molecule as claimed in claim 3 wherein said point mutationresults in a valine residue being substituted by an amino acid having amore bulky side chain and/or a β-carbon atom substituted only byhydrogen atoms.
 5. An isolated DNA molecule as claimed in claim 4wherein said point mutation results in a valine residue being replacedby methionine or leucine.
 6. An isolated DNA molecule as claimed inclaim 5 wherein said point mutation results in replacement of V287 inthe β2 subunit.
 7. An isolated DNA molecule as claimed in claim 6wherein said point mutation is a G to A nucleotide transition at base1025 in order to produce a V287M transition in the β2 subunit.
 8. Anisolated DNA molecule as claimed in claim 7 wherein the DNA moleculecomprises the nucleotide sequence set forth in SEQ ID NO:1.
 9. Anisolated DNA molecule as claimed in any one of claims 1 to 7 in whichone or more additional mutation events selected from the groupconsisting of point mutations, deletions, insertions and rearrangementshave occurred.
 10. An isolated DNA molecule as claimed in claim 9wherein said one or more additional mutation events are point mutationswhich result in conservative amino acid substitutions within theβ-subunit.
 11. An isolated DNA molecule comprising the nucleotidesequence set forth in SEQ ID NO:1.
 12. An isolated DNA moleculeconsisting of the nucleotide sequence set forth in SEQ ID NO:1.
 13. Anisolated polypeptide, said polypeptide being a mutant β-subunit of amammalian nicotinic acetylcholine receptor (nAchR), wherein a mutationevent selected from the group consisting of substitutions, deletions,insertions and rearrangements has occurred in the M2 domain and saidmutation event disrupts the functioning of an assembled mammaliannicotinic acetylcholine receptor so as to produce an epilepsy phenotype.14. An isolated polypeptide as claimed in claim 13 wherein said mutationevent is a substitution.
 15. An isolated polypeptide as claimed in claim14 wherein there is substitution of a valine residue.
 16. An isolatedpolypeptide as claimed in claim 15 wherein said valine residue issubstituted by an amino acid having a more bulky side chain and/or aβ-carbon atom substituted only by hydrogen atoms.
 17. An isolatedpolypeptide as claimed in claim 16 wherein said valine residue isreplaced by methionine or leucine.
 18. An isolated polypeptide asclaimed in claim 17 wherein said valine is V287 in the β2 subunit. 19.An isolated polypeptide as claimed in claim 18 wherein the substitutionis a V287M transition in the β2 subunit.
 20. An isolated polypeptide asclaimed in claim 19 comprising the amino acid sequence set forth in SEQID NO:2.
 21. An isolated polypeptide as claimed in claims 13 to 19 inwhich one or more additional mutation events selected from the groupconsisting of substitutions, deletions, insertions and rearrangementshave occurred.
 22. An isolated polypeptide as claimed in claim 21wherein said one or more mutation events are conservative substitutions.23. An isolated polypeptide comprising the amino acid sequence set forthin SEQ ID NO:2.
 24. An isolated polypeptide consisting of the amino acidsequence set forth in SEQ ID NO:2.
 25. An isolated polypeptide, saidpolypeptide being an assembled mammalian nicotinic acetylcholinereceptor, comprising at least one α-subunit and at least one β-subunit,wherein a mutation event selected from the group consisting ofsubstitutions, deletions, insertions and rearrangements has occurred inthe M2 domain of a β-subunit and said mutation event disrupts thefunctioning of said assembled mammalian nicotinic acetylcholine receptorso as to produce an epilepsy phenotype.
 26. An isolated polypeptide asclaimed in claim 25 wherein said mutation event is a substitution. 27.An isolated polypeptide as claimed in claim 26 wherein there issubstitution of a valine residue.
 28. An isolated polypeptide as claimedin claim 27 wherein said valine residue is substituted by an amino acidhaving a more bulky side chain and/or a β-carbon atom substituted onlyby hydrogen atoms.
 29. An isolated polypeptide as claimed in claim 28wherein said valine residue is replaced by methionine or leucine.
 30. Anisolated polypeptide as claimed in claim 29 wherein said valine is V287in the β2 subunit.
 31. An isolated polypeptide as claimed in claim 30wherein the substitution is a V287M transition in the β2 subunit.
 32. Anisolated polypeptide as claimed in claim 31 wherein the β2 subunitcomprises the amino acid sequence set forth in SEQ ID NO:2.
 33. Anisolated polypeptide as claimed in claims 25 to 32 in which one or moreadditional mutation events selected from the group consisting ofsubstitutions, deletions, insertions and rearrangements have occurred.34. An isolated polypeptide as claimed in claim 33 wherein said one ormore mutation events are conservative substitutions.
 35. An isolatedpolypeptide as claimed in any one of claims 25 to 34 wherein a furthermutation event selected from the group consisting of substitutions,deletions, insertions and rearrangements has occurred in the M2 domainof at least one further β-subunit, and said further mutation eventindependently disrupts the functioning of said assembled mammaliannicotinic acetylcholine receptor so as to produce an epilepsy phenotype.36. An isolated polypeptide as claimed in claim 35 wherein saidassembled nicotinic acetylcholine receptor comprises a plurality of β2subunits, and an identical mutation event has occurred in any one or allof these.
 37. An isolated polypeptide as claimed in claim 36 whereinsaid assembled nicotinic acetylcholine receptor consists of three β2subunits with a V287M mutation in any one or all of these, and two α4subunits.
 38. A method preparing a polypeptide, said polypeptide being amutant β-subunit of a mammalian nicotinic acetylcholine receptor,comprising the steps of: (1) culturing host cells transfected with anexpression vector comprising a DNA molecule as claimed in any one ofclaims 1 to 12 under conditions effective for polypeptide productions;and (2) harvesting the mutant β-subunit.
 39. A method as claimed inclaim 38 further comprising the step of allowing the mutant β-subunitand other subunits of the mammalian nicotinic acetylcholine receptor toassemble into a mammalian nicotinic acetylcholine receptor andharvesting the assembled receptor.
 40. An antibody which isimmunologically reactive with a polypeptide as defined in any one ofclaims 13 to 37, but not with a wild-type nicotinic acetylcholinereceptor or subunit thereof.
 41. An antibody as claimed in claim 40which is a monoclonal antibody.
 42. A method of treating epilepsy,comprising administering a selective antagonist of the nicotinicacetylcholine receptor when it contains a mutation in the M2 domain of aβ-subunit, said mutation being causative of epilepsy, to a subject inneed of such treatment.
 43. A method as claimed in claim 42 wherein theselective antagonist is an antibody.
 44. A method as claimed in claim 43wherein the antibody is a monoclonal antibody.
 45. A method as claimedin any one of claims 42 to 44, further comprising the step ofintroducing a wild-type nicotinic acetylcholine receptor to saidsubject.
 46. A method as claimed in claim 45 wherein the wild-typenicotinic acetylcholine receptor is introduced by gene therapy.
 47. Amethod as claimed in claim 45 wherein the wild-type nicotinicacetylcholine receptor is introduced by administering a substantiallypurified wild-type nicotinic acetylcholine receptor or nicotinicacetylcholine receptor β-subunit polypeptide.
 48. The use of a selectiveantagonist of the nicotinic acetylcholine receptor when it contains amutation in the M2 domain of a β-subunit, said mutation being causativeof epilepsy, in the manufacture of a medicament for the treatment ofepilepsy.
 49. A method of treating epilepsy, comprising administering anisolated DNA molecule which is the complement of any one of the DNAmolecules defined in claims 1 to 12 and which encodes a mRNA thathybridises with the mRNA encoding the β-subunit of the nicotinicacetylcholine receptor when it contains a mutation causative of epilepsyin the M2 domain, to a subject in need of such treatment.
 50. A methodas claimed in claim 49, further comprising the step of introducing awild-type nicotinic acetylcholine receptor to said subject.
 51. The useof an isolated DNA molecule which is a complement of a DNA molecule asdefined in any one of claims 1 to 12 and which encodes a mRNA thathybridises with the mRNA encoding the β-subunit of the nicotinicacetylcholine receptor when it contains a mutation causative of epilepsyin the M2 domain, in the manufacture of a medicament for the treatmentof epilepsy.
 52. The use of an isolated DNA molecule as claimed in anyone of claims 1 to 12 for the diagnosis of epilepsy.
 53. The use of apolypeptide as defined in any one of claims 13 to 37 in the diagnosis ofepilepsy.
 54. The use of an antibody as claimed in either claim 40 or 41in the diagnosis of epilepsy.
 55. A method for the diagnosis ofepilepsy, comprising the steps of: (1) obtaining DNA from a subjectsuspected of epilepsy; and (2) comparing the DNA sequence of a β-subunitof the nicotinic acetylcholine receptor of said DNA to the DNA sequenceof the corresponding β-subunit of the wild-type nicotinic acetylcholinereceptor.
 56. A method as claimed in claim 55 wherein each DNA fragmentis sequenced and the sequences compared.
 57. A method as claimed inclaim 55 wherein the DNA fragments are subjected to restriction enzymeanalysis.
 58. A method as claimed in claim 55 wherein the DNA fragmentsare subjected to SSCP analysis.
 59. A method for the diagnosis ofepilepsy, comprising the steps of: (1) obtaining the nicotinicacetylcholine receptor from a subject suspected of epilepsy; and (2)comparing a β-subunit of said receptor with the corresponding β-subunitof the wild-type nicotinic acetylcholine receptor.
 60. Use of apolypeptide as defined in any one of claims 13 to 37 in the screening ofcandidate pharmaceutical agents.
 61. Use as claimed in claim 60 whereinhigh-throughput screening techniques are employed.
 62. A geneticallymodified non-human animal transformed with an isolated DNA molecule asdefined in any one of claims 1 to
 12. 63. A genetically modifiednon-human animal as claimed in claim 62 in which the animal is selectedfrom the group consisting of rats, mice, hamsters, guinea pigs, rabbits,dogs, cats, goats, sheep, pigs and non-human primates such as monkeysand chimpanzees.
 64. A genetically modified non-human animal as claimedin claim 63 wherein the animal is a mouse.
 65. The use of a geneticallymodified non-human animal as claimed in any one of claims 62 to 64 inthe screening of candidate pharmaceutical compounds.
 66. The use of acell transformed with a DNA molecule as claimed in any one of claims 1to 12 in the screening of candidate pharmaceuticals.
 67. A host celltransformed with a DNA molecule as claimed in any one of claims 1 to 12.68. An expression vector comprising a DNA molecule as claimed in any oneof claims 1 to 12.